Compound as cholinesterase inhibitor and its isolation from fungus sporotrichum species

ABSTRACT

The present invention provides a novel bioactive compound 12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE “Sporotricolone”, mainly as acetylcholinesterase (AchE) inhibitor, along with a process for the isolation of said compound from fungus Sporotrichum species.

FIELD OF INVENTION

[0001] The present invention relates to a compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone”, mainly as acetylcholinesterase (AchE) inhibitor. Thepresent invention also relates to a process for the isolation of saidcompound from fungus Sporotrichum species.

BACKGROUND AND PRIOR ART REFERENCES

[0002] Enzyme inhibitors are important class of molecules that are usedas drugs and pesticides. The enzyme acetylcholinesterase (AchE) isinvolved in the synaptic transmission of the nerve impulse and itsinhibition leads to accumulation of the neurotransmitter, acetylcholineleading to overexcitation of the postsynaptic neuron. This property ofthe inhibitor has been exploited to develop newer insecticides against awide range of insect pests as well as drugs effective against worms,and, recently a new class of neuroactive drugs against dementia(Alzheimer's).

[0003] Although earlier authors have isolated metabolites such asastericacis, questin and questinol from Sorortricum sp., no AchE inhibitoractivity has been reported(Slater, G P, Haskins, R H and Hogge, L. R.Can J Mirobiol 17 (1971), 1576-79). The fungi, Aspergillus terreus (Ling, K H, Liou, H H, Yang, C M and Yang C K, Appl. Env. Microbiol, 37(1979) 355-57) and Penicillium sp. (Omura, Skuno, F, Otoguro, K, Shiomi,K. Mauma, R and Iwai, Y. J. Antibiot. 48(1995) 745-46 ) have beenreported to produce an AchE inhibitor named Arusigacin. However, theAchE inhibitor of the present invention is isolated from Sporotrichumhaving distinct chemical structure and properties and therefore a novelinhibitor molecule.

[0004] After screening of various microorganisms, a fungal culture isselected which shows inhibition against a serineesterase/protease/cholinesterase enzyme. This imperfect deuteromycetes,Sporotrichum species and was first isolated in 1966. The taxonomicfeatures of Sporotrichum species (deuteromyces) are broad hyphae andseptate in nature; has hyalline conidiophores with littledifferentiation from vegetatative hyphae and solitary conidia with broadattachment to the hyphae.

[0005] This culture has previously been a subject of researchinvestigation at the Central Food Technological Research Institute(CFTRI) India, for its ability to grow on lignocellulosic wastes for theproduction of enzymes and organic acids (Sreekantaiah, K R, PhD thesis(1976) University of Mysore; Manonmani, H K, PhD thesis (1986)University of Mysore).

[0006] This culture has now been used in the present invention toproduce a fermented extract containing a serineesterase/protease/cholinesterase inhibitor.

[0007] The conditions of fermentation have been described earlier inIndian Patent Application No. 303/DEL/2000 Sattur, A P, Shivanandappa,T, Divakar, S and Karanth, N G.

OBJECTS OF THE INVENTION

[0008] The main object of the present invention is to provide bioactivecompound 12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone” having inhibitory activity against acetylcholinesterase(AchE).

[0009] Another object of the present invention is to provide a compoundhaving inhibitory activity against serine esterase and protease.

[0010] Yet another object of the present invention is to provide acompound having insecticidal properties.

[0011] Still another object of the present invention is to provide acompound having enhanced cholinergic activity.

[0012] Yet another object of the present invention is to provide aprocess for the isolation of Sporotricolone.

SUMMARY OF THE INVENTION

[0013] The present invention provides a compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone” mainly as acetylcholinesterase (AchE) inhibitor. Thepresent invention also provides a process for the isolation of saidcompound from fungus Sporotrichum species.

DETAILED DESCRIPTION OF THE INVENTION

[0014] Accordingly, the present invention provides a bioactive compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone” of formula (I) having acetylcholinesterase (AchE)enzyme inhibition activity obtained from fungus Sporotrichum species.

[0015] An embodiment of the present invention wherein the compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4ONE“Sporotricolone” of formula (I) having the following characteristicproperties:

[0016] Solubility: Highly soluble in ethyl acetate, methanol andacetone. UV (ethyl acetate) λ_(max): 265 nm, 312 nm. ¹HNMR spectrum(DMSO, δ_(TMS=)0.00 ppm); δ 1.03 (3H, d, J=6.3 Hz, —CH—CH ₃) δ 1.2-2.7(14H, m, 7× —CH ₂) δ 3.6-3.8 (m, Ar—O—CH ₃ and Ar—CH—OH) δ 7.20 (1H, d,J=2.5 Hz, C_(6′)—H) δ 7.30 (2H, d, J=7.1 Hz, C_(3′)—Hand C_(4′)—H) Massspectrum (EI, 70 eV, 25° C., 200-ul amp): m/e: 336 (M+), 279(366-87),167(274-112), 57 (CH₂COCH₃), 43, 29.

[0017] Another embodiment of the present invention, wherein the purityof the compound is established by TLC and RP HPLC.

[0018] Yet another embodiment of the present invention, wherein saidcompound is named as 12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE “Sporotricolone”.

[0019] Still another embodiment of the present invention, wherein saidcompound is an inhibitor of the enzyme acetylcholinesterase from the ratbrain as well as erythrocytes with a IC₅₀ value of 20×10⁻⁶ M.

[0020] Yet another embodiment of the present invention, wherein saidcompound also acts as an inhibitor of serine esterase of the rat liverserum.

[0021] Still another embodiment of the present invention, wherein saidcompound having insecticidal properties.

[0022] Yet another embodiment of the present invention, wherein saidcompound effective against mosquito larvae at an optimum concentrationof 70 μg/ml water (70 ppm) when exposed for 24 hrs.

[0023] Still another embodiment of the present invention, wherein theinsecticidal activity of the compound against mosquito larvae isselected from culex quinquifasciatus.

[0024] Yet another embodiment of the present invention, wherein saidcompound as acetylcholineesterase inhibitor having potential applicationas a drug for Alzheimer's disease or dermentia.

[0025] The present invention also provides a process for the isolationof 12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4ONESporotricolone from the fungus Sporotrichum species, said processcomprising the steps of:

[0026] (a) extracting the fermented solid with an organic solvent;

[0027] (b) filtering the extract of step (a) through a cloth or Whatmanfilter paper to obtain a clear solution;

[0028] (c) evaporating the solution of step (b) under reduced pressureto obtain a crude extract;

[0029] (d) purifying the crude extract of step (c) by columnchromatography over silica gel and eluting with mixture of organicsolvents of increasing polarity;

[0030] (e) pooling active eluted fraction of step (d) and furthersubjected to column chromatography over silica gel by eluting withmixture of organic solvents with increasing polarity;

[0031] (f) repooling the active eluted fractions of step (e);

[0032] (g) evaporating the pooled fractions of step (f) to get aresidue; and

[0033] (h) dissolving the residue in step (g) in ethyl acetate to yieldthe pure compound “Sporotricolone”.

[0034] Yet another embodiment of the present invention, a processwherein in step (a) the organic solvent is selected from a groupconsisting of ethyl acetate, acetone or methanol and preferably ethylacetate.

[0035] Still another embodiment of the present invention, a processwherein in step (d) the mixture of organic solvents is selected from thecombination of hexane:diethyl ether and chloroform:methanol mixtures.

[0036] Further embodiment of the present invention, a process wherein instep (e) the mixture of organic solvent used is chloroform:ethyl acetatemixture.

[0037] Yet another embodiment of the present invention, wherein the saidcompound is separated and purified by column chromatography on silicagel and RP HPLC.

[0038] Still another embodiment of the present invention, wherein saidcompound having an UV absorption at 265 and 312 nm.

[0039] The present invention is further explained in the form offollowing embodiments

[0040] In the present invention a process for the isolation of anacetylcholinesterase inhibitor, which comprises the extraction of thefermented broth culture with solvents such as ethyl acetate. The crudeextract is further extracted with 10-20 ml of methanol and subjected tocolumn chromatography using silica gel and eluted with variouscombinations of solvents such as hexane:diethyl ether (85:15, 50:50)followed by chloroform:methanol (95:5, 50:50, 10:90). Fractions areevaporated under nitrogen, dissolved in ethyl acetate and assayed foracetylcholinesterase (AchE) inhibition. The active fractions are pooledand further subjected to purification on silica gel columnchromatography and eluted with chloroform:ethyl acetate (90:10, 50:50,0:100). The active fractions pooled and the solvent evaporated anddissolved in 2 ml ethyl acetate. The purity, as checked by TLC, showed asingle spot and HPLC on reverse phase column (C18) with chloroform andmethanol as mobile phase. The yield is about 10 mg. The purifiedinhibitor showed inhibitor potency against rat brain AchE with an IC₅₀of 15-20×10⁻⁶ M.

[0041] A general process for the production of the novelAcetylcholinesterase inhibitor is given in the flow sheet:

[0042] The structure of the isolated inhibitor is determined by UV, ¹HNMR and mass spectrometry.

[0043] The following examples are given by way of illustration of thepresent invention and should not be construed to limit the scope of theinvention.

EXAMPLE-1

[0044] The fermentation culture is extracted with 100 ml of ethylacetate, filtered through a cotton filter and concentrated in vacuo toobtain 1 ml of the crude extract. This is used as a source of the enzymeinhibitor and 20 μl of the extract gave 60-90% inhibition of rat brainacetylcholinesterase enzyme.

EXAMPLE-2

[0045] The crude extract is further extracted with 10-20 ml of methanoland subjected to column chromatography using silica gel and eluted withvarious combinations of solvents such as hexane:diethyl ether (85:15,50:50) followed by chloroform:methanol (95:5, 50:50, 10:90). Fractionsare evaporated under nitrogen, dissolved in ethyl acetate and assayedfor acetyl cholinesterase (AchE) inhibitor. The active fractions(#11-19) are pooled and further subjected to purification on silica gelcolumn chromatography and eluted with chloroform:ethyl acetate (90:10,50:50, 0:100). The active fractions pooled and the solvent evaporatedand dissolved in 2 ml ethyl acetate. The purity, as checked by TLC,showed a single spot. RP HPLC also ascertained the purity on a C18column with chloroform and methanol as the mobile phase wherein it is asingle peak. The yield is about 10 mg.

EXAMPLE-3

[0046] The purified inhibitor showed inhibitor potency against rat brainAchE with an IC50 of 15-20×10⁻⁶ M, as assayed according to Ellman etal., (Biochem. Pharmacol. 7(1961), 88-95) and is given as follows: Theenzyme inhibition is carried out by pre-incubating the enzyme (rat brainacetylcholinesterase) with 2-20 ul of the culture extract or the columnfraction at room temperature (25° C.) for 15 minutes followed by theaddition of the substrate, acetyl thiocholine iodide (0.5 mM), in 3 mlphosphate buffer (0.1.M, pH.7.4) containing 0.25 mMdithiobisnitrobenzoic acid. Absorbance change at 412 nm is monitoredevery 30 seconds for 2 min in an UV-VIS Spectrophotometer. Inhibition iscalculated relative to the solvent control. IC₅₀ is determined byregression analysis.

Advantages

[0047] 1. The present invention provides an AchE /serineesterase/protease inhibitor from a microbial source.

[0048] 2. The present invention provides a simple extraction andchromatographic procedure to purify the AchE inhibitor from the crudemixture.

[0049] 3. In the present invention the isolated inhibitor is a novelbioactive molecule

1. A bioactive compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12DIHYDROXY-DODECA-4-ONE“Sporotricolone” of formula (I) having primarily acetylcholinesterase(AchE) enzyme inhibitor activity that is obtained from fungusSporotrichum species.


2. The compound according to claim 1 having the following characteristicproperties: Solubility: Highly soluble in ethyl acetate, methanol andacetone. UV (ethyl acetate) λ_(max): 265 nm, 312 nm. ¹HNMR spectrum(DMSO, δ_(TMS=)0.00 ppm); δ 1.03 (3H, d, J=6.3 Hz, —CH—CH ₃) δ 1.2-2.7(14H, m, 7× —CH ₂) δ 3.6-3.8 (m, Ar—O—CH ₃ and Ar—CH—OH) δ 7.20 (1H, d,J=2.5 Hz, C_(6′)—H) δ 7.30 (2H, d, J=7.1 Hz, C_(3′)—Hand C_(4′)—H) Massspectrum (EI, 70 eV, 25° C., 200-ul amp): m/e: 336 (M+), 279(366-87),167(274-112), 57 (CH₂COCH₃), 43,
 29. 3. The compound according to claim1, wherein the purity of the compound is established by TLC and RP HPLC.4. The compound according to claim 1, wherein said compound is named as“Sporotricolone”.
 5. The compound according to claim 1, wherein saidcompound is an inhibitor of the enzyme acetylcholinesterase from the ratbrain as well as erythrocytes with a IC₅₀ value of 20×10⁻⁶ M.
 6. Thecompound according to claim 1, wherein said compound also acts as aninhibitor of serine esterase of the rat liver serum.
 7. The compoundaccording to claim 1, wherein said compound having insecticidalproperties.
 8. The compound according to claim 1, wherein said compoundeffective against mosquito larvae at an optimum concentration of 70μg/ml water (70 ppm) when exposed for 24 hrs.
 9. The compound accordingto claim 1, wherein the insecticidal activity of the compound againstmosquito larvae is selected from Culex quinquifasciatus.
 10. Thecompound according to claim 1, wherein said compound asacetylcholineesterase inhibitor having potential application as a drugfor Alzheimer's disease or dementia.
 11. A process for the isolation ofthe compound12-(2′-CARBOXY-5′-METHOXYPHENYL)-2,12-DIHYDROXY-DODECA-4-ONE“Sporotricolone” from the fungus Sporotrichum species, said processcomprising the steps of: (a) extracting the fermented solid with anorganic solvent; (b) filtering the extract of step (a) through a clothor Whatman filter paper to obtain a clear solution; (c) evaporating thesolution of step (b) under reduced pressure to obtain a crude extract;(d) purifying the crude extract of step (c) by column chromatographyover silica gel and eluting with mixture of organic solvents ofincreasing polarity; (e) pooling active eluted fraction of step (d) andfurther subjected to column chromatography over silica gel by elutingwith mixture of organic solvents with increasing polarity; (f) repoolingthe active eluted fractions of step (e); (g) evaporating the pooledfractions of step (f) to get a residue; and (h) dissolving the residuein step (g) in ethyl acetate to yield the pure compound“Sporotricolone”.
 12. The process according to claim 11, wherein in step(a) the organic solvent is selected from a group consisting of ethylacetate, acetone or methanol and preferably ethyl acetate.
 13. Theprocess according to claim 11, wherein in step (d) the mixture oforganic solvents is selected from the combination of hexane:diethylether and chloroform:methanol mixtures.
 14. The process according toclaim 11, wherein in step (e) the mixture of organic solvent used ischloroform:ethyl acetate mixture.